The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity

Article

The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity Graphical Abstract

Authors Zhe Huang, Ling Zhong, Jimmy Tsz Hang Lee, ..., Yu Wang, Chi-Ming Wong, Aimin Xu

Correspondence [email protected] (C.-M.W.), [email protected] (A.X.)

In Brief Zhe Huang et al. show that cold activates type 2 immune responses and beiging in subcutaneous WAT through an FGF21CCL11 axis which drives eosinophil recruitment, M2 macrophage accumulation and proliferation and commitment of beige adipocyte precursors. These findings explain how the immune system communicates with sympathetic nerves to control adaptive thermogenesis.

Highlights d

Cold exposure selectively induces FGF21 expression in thermogenic adipose tissues

d

Autocrine actions of FGF21 are obligatory for type 2 immune activation and beiging

d

FGF21 induces CCL11 production in adipocytes to promote recruitment of eosinophils

d

The FGF21-CCL11 axis couples nervous and immune systems to induce beiging in scWAT

Huang et al., 2017, Cell Metabolism 26, 1–16 September 5, 2017 ª 2017 Elsevier Inc. http://dx.doi.org/10.1016/j.cmet.2017.08.003

Please cite this article in press as: Huang et al., The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity, Cell Metabolism (2017), http://dx.doi.org/10.1016/j.cmet.2017.08.003

Cell Metabolism

Article The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity Zhe Huang,1,2 Ling Zhong,1,2 Jimmy Tsz Hang Lee,1,2 Jialiang Zhang,1,2 Donghai Wu,4 Leiluo Geng,1,2 Yu Wang,1,3 Chi-Ming Wong,1,2,3,* and Aimin Xu1,2,3,5,* 1The State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Kong, 21 Sassoon Road, Laboratory Block, Pokfulam, Hong Kong, China 2Department of Medicine, The University of Hong Kong, Hong Kong, China 3Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, China 4The Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China 5Lead Contact *Correspondence: [email protected] (C.-M.W.), [email protected] (A.X.) http://dx.doi.org/10.1016/j.cmet.2017.08.003

SUMMARY

Type 2 cytokines are important signals triggering biogenesis of thermogenic beige adipocytes in white adipose tissue (WAT) during cold acclimation. However, how cold activates type 2 immunity in WAT remains obscure. Here we show that cold-induced type 2 immune responses and beiging in subcutaneous WAT (scWAT) are abrogated in mice with adipose-selective ablation of FGF21 or its co-receptor b-Klotho, whereas such impairments are reversed by replenishment with chemokine CCL11. Mechanistically, FGF21 acts on adipocytes in an autocrine manner to promote the expression and secretion of CCL11 via activation of ERK1/2, which drives recruitment of eosinophils into scWAT, leading to increases in accumulation of M2 macrophages, and proliferation and commitment of adipocyte precursors into beige adipocytes. These FGF21-elicited type 2 immune responses and beiging are blocked by CCL11 neutralization. Thus, the adipose-derived FGF21CCL11 axis triggers cold-induced beiging and thermogenesis by coupling sympathetic nervous system to activation of type 2 immunity in scWAT.

INTRODUCTION While white adipocytes store energy in the form of triglycerides, brown adipocytes dissipate energy as heat due to the presence of uncoupling protein-1 (UCP1), a unique mitochondrial inner membrane protein which uncouples oxidative phosphorylation from ATP synthesis (Nedergaard et al., 2001). Besides classical brown adipocytes which are clustered in well-defined depots of brown adipose tissues (BATs, such as interscapular and axillary), there also exists a subset of inducible brown-like adipocytes (namely beige adipocytes) scattered within white adipose tissues (WATs) (Wu et al., 2012), which are activated

upon thermogenic stimuli such as cold exposure and physical exercise. A growing body of evidence from both animal and human studies suggests that activation of beige adipocytes represents a promising therapeutic strategy for obesity and its related cardiometabolic complications (Chang et al., 2012; Hanssen et al., 2015). Although beige adipocytes functionally and morphologically resemble classical brown adipocytes, they arise from different precursor cells and are activated by overlapping, but distinct mechanisms. Cold exposure-induced activation of classical brown adipocytes in BAT depots is mediated predominantly by norepinephrine released from the sympathetic nerve terminals, whereas beiging of WAT, which is much less innervated, is heavily dependent on its local microenvironment (Pope et al., 2016). In particular, WAT is home to an abundance of immune cells. Recent studies have uncovered an indispensable role of several type 2 immune cells and their related type 2 cytokines in promoting beiging of WAT (Brestoff et al., 2015; Hui et al., 2015; Lee et al., 2015; Qiu et al., 2014; Rao et al., 2014). Upon cold exposure, eosinophils are rapidly recruited into WAT, thereby inducing polarization of alternatively activated M2 macrophages through production of interleukin-4 (IL-4). IL-4 in turn stimulates M2 macrophages to produce norepinephrine by activation of tyrosine hydroxylase, thus enhancing thermogenesis and adipogenesis of beige adipocytes in WAT (Qiu et al., 2014; Rao et al., 2014; Wu et al., 2011). In addition, IL-4 acts directly on Lin
CD31– CD45– Sca-1+ PDGFRa+ adipocyte precursors (APs) to induce beige adipogenesis (Lee et al., 2015; Uhm and Saltiel, 2015). Type 2 innate lymphoid cells (ILC2s), which reside in subcutaneous WAT (scWAT) of both mice and humans, play important roles in maintaining the presence of eosinophils and M2 macrophages in the adipose depots (Lee et al., 2015; Molofsky et al., 2013). Furthermore, ILC2s promote beige adipogenesis by secreting methionine-enkephalin peptides, which can induce UCP1 expression in WAT (Brestoff et al., 2015). However, the physiological signals that mediate cold challenge-induced activation of these type 2 immune responses and adaptive thermogenesis in WAT still remain obscure. Fibroblast growth factor 21 (FGF21), a member of the endocrine FGF subfamily, has pleiotropic effects on the regulation Cell Metabolism 26, 1–16, September 5, 2017 ª 2017 Elsevier Inc. 1

Please cite this article in press as: Huang et al., The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity, Cell Metabolism (2017), http://dx.doi.org/10.1016/j.cmet.2017.08.003

of energy homeostasis and insulin sensitivity via binding with its receptor complex between FGF receptor-1 (FGFR1) and co-receptor b-Klotho (KLB) (Fisher and Maratos-Flier, 2016). Chronic administration of pharmacological doses of FGF21 leads to a plethora of therapeutic benefits for obesity-related cardiometabolic complications, including reduction of fat mass, alleviation of hyperglycemia, insulin resistance, dyslipidemia, atherosclerosis, and diabetic cardiomyopathy (Kharitonenkov and DiMarchi, 2015). Physiologically, FGF21 is a stressresponsive hormone and its expression is markedly induced by a diverse range of physiological or pathological stresses, such as starvation, nutrient excess, mitochondrial stress, drug toxicity, exercise, and cold exposure (Kim and Lee, 2014). The induction of FGF21 by these stimuli serves as an adaptive mechanism to defend against excessive stress. Although the liver is the main contributor to circulating FGF21, adipose tissues are also important organs for both production and actions of FGF21 (Fisher and Maratos-Flier, 2016). In adipocytes, FGF21 enhances insulin-independent glucose uptake, promotes mitochondrial oxidation, and induces secretion of adiponectin, which in turn mediates the glucose-lowering and insulin-sensitizing effects of FGF21 (Chau et al., 2010; Holland et al., 2013; Kharitonenkov et al., 2005; Lin et al., 2013). Furthermore, the peroxisome proliferator-activated receptor g (PPARg) agonists thiazolidinediones (TZDs) induce expression and secretion of FGF21 in adipocytes, and the therapeutic benefits of TZDs are mediated in part by FGF21 actions in adipocytes, where it functions in a feedforward loop to modulate PPARg activity (Dutchak et al., 2012). In contrast, another parallel study shows that FGF21 is not required for the antidiabetic actions of TZDs (Adams et al., 2013). Instead, TZDs enhance FGF21 sensitivity by upregulation of its co-receptor KLB (Kharitonenkov et al., 2008; Moyers et al., 2007). In response to cold challenge, FGF21 is induced selectively in adipose tissues, but not in liver (Fisher et al., 2012; Hondares et al., 2011). Global deletion of FGF21 in mice leads to impairments in cold-induced beiging of scWAT, while administration of recombinant FGF21 increases beiging of scWAT and total energy expenditure in mice (Fisher et al., 2012). However, it is currently unclear whether coldinduced beiging of scWAT is mediated by autocrine or endocrine actions of FGF21. The molecular mechanisms whereby FGF21 modulates beiging and adaptive thermogenesis in scWAT remain poorly defined. In this study, by using a series of tissue-selective FGF21 and KLB knockout (KO) mice, we demonstrated that the autocrine action of FGF21 in mature adipocytes is an obligatory early step in driving cold-induced beige adipocyte biogenesis by activating type 2 immunity in scWAT. Furthermore, we identified the chemoattractant C-C motif chemokine ligand 11 (CCL11) as a downstream responder of FGF21 in mature adipocytes, which in turn triggers type 2 immune responses and beige adipogenesis by recruitment of eosinophils. RESULTS FGF21 Expression Is Selectively Induced in Thermogenic Adipose Depots by Cold Exposure To explore the physiological roles of FGF21 in cold-induced adaptive thermogenesis, we first measured the dynamic changes 2 Cell Metabolism 26, 1–16, September 5, 2017

of FGF21 expression in various types of adipose tissues (scWAT, BAT, and epididymal WAT [eWAT]), the liver, and the circulation of C57BL/6J male mice exposed to cold (6 C) for 6 days after being housed at thermoneutral condition (30 C) for 2 weeks (Figure 1). Quantitative real-time PCR analysis revealed a progressive increase in the expression of Fgf21 mRNA in scWAT and BAT after cold exposure (Figures 1A and 1B), but not in eWAT (Figure 1C) or liver (Figure 1D), which was consistent with changes in the protein concentrations of FGF21 in these tissues (Figures 1E–1H). Furthermore, the magnitude of induction in the expression of Fgf21 in scWAT (30-fold on day 1 and >60-fold on days 3 and 6 after cold exposure) was higher and more persistent than that in BAT (maximal induction by 6- to 7-fold on day 1 post-exposure, and declined to 3- to 4-fold on days 3 and 6). Further cell fractional analysis showed that cold-induced Fgf21 expression occurred predominantly in mature adipocytes, but not in the stromal vascular fraction (SVF) (Figure 1I). However, consistent with previous studies (Fisher et al., 2012), there was no obvious change in the serum level of FGF21 throughout the period of cold exposure (Figure 1J). Although there was a mild increase in serum levels of FGF21 after 6 hr and 1 day of cold exposure, the change did not reach statistical significance (Figure 1J). As expected, the expression of FGF21 receptor complex (Klb and Fgfr1) was predominantly enriched in mature adipocytes, but not SVF (Figures 1K and 1L). Cold exposure had no obvious effect on expression of either Klb or Fgfr1 in both fractions. Taken together, our data demonstrate a selective induction of Fgf21 expression, but not its receptor complex, in thermogenic adipose depots (scWAT and BAT), but not in eWAT, liver, or circulation. Adipose-Specific FGF21 Deficiency Leads to Impaired Adaptation to Cold Environment in Mice To further dissect the contribution of adipose- and liver-derived FGF21 in cold-induced thermogenesis, we generated both adipose tissue-specific FGF21 KO (A-FGF21KO) and liver-specific FGF21 KO (L-FGF21KO) mice by crossing mice bearing a conditional Fgf21 allele with exons 1 and 2 floxed (FGF21lox/lox) with aP2-Cre and albumin-Cre transgenic mice, respectively (Figure S1A). The genotypes of both mouse strains were confirmed by PCR (Figure S1B) and real-time PCR analyses, showing selective ablation of Fgf21 expression in adipose tissues in A-FGF21KO mice and in liver in L-FGF21KO mice (Figures S1C and S1D). As expected, both hepatic (Figure S1E) and circulating (Figure S1F) levels of FGF21 were markedly induced by fasting in FGF21lox/lox (control) mice and A-FGF21KO mice (Figures S1E and S1F). However, this fasting-induced FGF21 elevation in both liver and serum was negligible in L-FGF21KO mice (Figures S1E and S1F), confirming that circulating FGF21 is derived predominantly from liver (Markan et al., 2014). Both A-FGF21KO and L-FGF21KO mice developed and grew normally as previously reported (Markan et al., 2014). Both A-FGF21KO mice and their control littermates had a similar rectal temperature under the thermoneutral condition and early time points (within 6 hr) after cold challenge at 6 C (Figure 2A). However, A-FGF21KO mice showed a significantly lower rectal temperature (by 1 C) than control mice after prolonged cold challenge (Figure 2A), suggesting adipose-derived FGF21 was required for maintaining whole-body temperature by non-shivering adaptive thermogenesis. To further investigate

Please cite this article in press as: Huang et al., The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity, Cell Metabolism (2017), http://dx.doi.org/10.1016/j.cmet.2017.08.003

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Figure 1. Cold Exposure Markedly Induces the Expression of FGF21, but Not Its Receptors in Thermogenic Adipose Tissues Twelve-week-old male C57BL/6J mice were housed at thermoneutral condition (30 C) for 2 weeks before cold exposure at 6 C for various time periods as indicated. (A–D) The mRNA expression of Fgf21 in inguinal subcutaneous WAT (scWAT) (A), interscapular BAT (B), epididymal WAT (eWAT) (C), and liver (D), as determined by real-time PCR analysis. (E–H) The protein concentration of FGF21 in scWAT (E), BAT (F), eWAT (G), and liver (H) as determined by ELISA. (I) The relative mRNA abundance of the Fgf21 gene in stromal vascular fraction (SVF) and mature adipocyte (MA) fraction isolated from scWAT of mice housed at 30 C or 6 C for 6 days. (J) Serum levels of FGF21 as determined by ELISA. (K and L) The relative mRNA abundance of the Klb (K) and Fgfr1 (L) in SVF and MA fractions isolated from scWAT of mice housed at 30 C or 6 C for 6 days. Data represent mean ± SEM; n = 4–6 per group; repeated with three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001.

the impact of deletion of adipose-derived FGF21 on thermogenesis in scWAT and BAT, we monitored the ventral and dorsal temperatures at the inguinal region and the interscapular region,

respectively, using infrared imaging. There was no obvious difference in the local temperatures at either inguinal or interscapular regions between A-FGF21KO mice and their wild-type Cell Metabolism 26, 1–16, September 5, 2017 3

Please cite this article in press as: Huang et al., The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity, Cell Metabolism (2017), http://dx.doi.org/10.1016/j.cmet.2017.08.003

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Figure 2. A-FGF21KO Mice Exhibit Impaired Adaptive Thermogenesis and Beiging of scWAT during Chronic Cold Exposure Twelve-week-old male A-FGF21KO mice and their FGF21lox/lox (control) littermates housed at 30 C for 2 weeks were subjected to cold exposure at 6 C or continued to be housed at 30 C for 6 days. (A) Rectal temperature of mice measured at different time points after cold exposure. (legend continued on next page)

4 Cell Metabolism 26, 1–16, September 5, 2017

Please cite this article in press as: Huang et al., The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity, Cell Metabolism (2017), http://dx.doi.org/10.1016/j.cmet.2017.08.003

control littermates under the thermoneutral condition (Figure 2B). The temperatures at both regions in the control mice slightly dropped after cold exposure for 6 days. Notably, although both types of mice showed a similar interscapular temperature after cold exposure, the temperature at the inguinal region of A-FGF21KO mice dropped more significantly when compared with the control littermates (Figure 2B), suggesting impairment in cold-induced thermogenesis in scWAT of A-FGF21KO mice. Both A-FGF21KO mice and their control littermates had similar whole-body oxygen consumption (VO2) under the thermoneutral condition (Figure 2C), and cold exposure led to a marked elevation of VO2 in both types of mice. However, the magnitude of cold-induced VO2 in A-FGF21KO mice was significantly lower than in wild-type control littermates (Figure 2C), suggesting that adipose-derived FGF21 was involved in maintaining whole-body energy expenditure during cold exposure. As expected, the expression levels of the key thermogenic genes in scWAT, including Ucp1, Cidea, Elovl3, and Cpt1b, were significantly induced by chronic cold exposure in control mice (Figure 2D). However, the induction of these genes was significantly reduced by 2- to 6-fold in scWAT of A-FGF21KO mice as compared with the control group (Figure 2D). Consistently, A-FGF21KO mice also exhibited an obvious impairment in cold-induced UCP1 protein expression (Figure 2E), and a marked reduction in the formation of multilocular and UCP1+ beige adipocytes in scWAT (Figures 2F and 2G). The impaired cold-induced beiging of scWAT in A-FGF21KO mice was further evident in reduced mtDNA content (Figure 2H), decreased expression of the mitochondrial electron transport chain subunits (complex I-III) (Figure 2I), and lower basal and norepinephrine-stimulated oxygen consumption rate (Figure 2J) in scWAT from A-FGF21KO mice after 6-day cold exposure. Interestingly, there was no significant change in classical BAT of A-FGF21KO mice with respect to thermogenic gene expression (Figure S2A), brown adipocyte morphology (Figure S2C), UCP1 protein expression (Figures S2B and S2D), and mitochondrial content (Figures S2E and S2F) compared with the control group at both 30 C and 6 C. Furthermore, there was no obvious difference in rectal temperature between the L-FGF21KO mice and their FGF21lox/lox (control) littermates housed at either 30 C or 6 C (Figure S3A). Cold exposure-induced beiging of scWAT in L-FGF21KO mice was intact as demonstrated by comparable levels of cold-induced UCP1 protein expression (Figure S3B), adipose morphology (Figure S3C), and the number of UCP1+ adipocytes (Figure S3D) between the two types of

mice. Taken together, our findings demonstrate that adiposederived, but not hepatic FGF21, is required for cold-induced beiging and adaptive thermogenesis in scWAT. Cold-Induced Recruitment of Eosinophils into scWAT Is Impaired in A-FGF21KO Mice To investigate the mechanisms by which FGF21 regulates coldinduced beiging of scWAT, we first tested whether FGF21 directly promotes UCP1 induction in adipocytes. Treatment of mouse primary adipocytes differentiated from SVF of scWAT with recombinant mouse FGF21 (rmFGF21) protein for 48 hr resulted in a 2.3-fold increase in mRNA expression of Ucp1 (Figure 3A), which was dramatically lower than that in scWAT under cold challenge in vivo (Figure 2D). Furthermore, the protein expression of UCP1 in adipocytes after treatment with rmFGF21 was not detectable (Figure 3B), suggesting that the marked effects of FGF21 on beiging of scWAT is unlikely to be explained by the direct induction of UCP1 in adipocytes. Since pharmacological treatment with FGF21 has been shown to stimulate the expression and secretion of adiponectin in adipocytes (Holland et al., 2013; Lin et al., 2013), and adiponectin enhances cold-induced beiging of scWAT by promoting proliferation of M2 macrophages (Hui et al., 2015), we then investigated the possibility of adipose-derived FGF21 in enhancing beiging of scWAT via induction of adiponectin. However, cold exposure-induced mRNA expression of adiponectin (Adn) in scWAT was comparable between A-FGF21KO and control mice (Figure 3C), suggesting that induction of FGF21 and adiponectin was not causally related during cold exposure. Indeed, the pharmacological dose of rmFGF21 protein (1 mg/mL) used in previous studies to induce the production of adiponectin from mouse adipocytes is over 15 times higher than the physiological concentration of FGF21 in WAT even after cold exposure (approximately 65 ng/mL when taken the average tissue density of 0.92 for WAT into account) (Figure 1E). Given that adipose-selective depletion of FGF21 caused the impairments in cold-induced beiging of scWAT, but not activation of classical BAT, we hypothesized that FGF21 may modulate the microenvironment required for beiging of scWAT, particularly type 2 immune cells and their secreted cytokines. As expected, cold exposure induced the expression of the key type 2 cytokines (IL-4 and IL-13) essential for beiging of scWAT in control mice (Figure 3D). However, the effect of cold exposure on induction of IL-4 and IL-13 expression was markedly impaired in scWAT of A-FGF21KO mice (Figure 3D). As IL-4 production is

(B) Representative infrared images showing A-FGF21KO and control mice housed at 30 C or 6 C. The right panels are quantification of the mean surface temperature in the inguinal and interscapular regions above scWAT (top) and BAT (bottom), respectively. (C) Whole-body oxygen consumption (VO2) in A-FGF21KO and control mice during 24-hr light/dark cycles by comprehensive laboratory animal monitoring system analysis. Data were normalized with body weight (BW). (D) Real-time PCR analysis for mRNA expression of several thermogenic genes, including uncoupling protein 1 (Ucp1), cell death-inducing DNA fragmentation factor a (Cidea), elongation of very long chain fatty acids protein 3 (Elovl3), and carnitine palmitoyltransferase 1b (Cpt1b) in scWAT. (E) Western blot analysis for UCP1 protein expression in scWAT. The bottom panel is the densitometric analysis for the relative abundance of UCP1 normalized with b-Tubulin. (F and G) Representative images of scWAT stained with H&E (F) and with an anti-UCP1 antibody (G). Scale bar, 20 mm. (H) Quantification of mitochondrial DNA (mtDNA) normalized with genomic DNA (gDNA) in scWAT as determined by real-time PCR analysis. (I) Western blot analysis for proteins from four complexes in the electron transport chain (ETC) (CI, NDUFB8; CII, SDHB; CIII, UQCRC2; CV, ATP5A) in protein lysates from scWAT. (J) Basal and norepinephrine (NE)-stimulated oxygen consumption rate (OCR) in the explants of scWAT. Data represent mean ± SEM; n = 5–6 per group; repeated with three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S1–S3.

Cell Metabolism 26, 1–16, September 5, 2017 5

Please cite this article in press as: Huang et al., The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity, Cell Metabolism (2017), http://dx.doi.org/10.1016/j.cmet.2017.08.003

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6 Cell Metabolism 26, 1–16, September 5, 2017

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mostly associated with eosinophils and ILC2s are the major sources of IL-13 in adipose tissues (Molofsky et al., 2013; Qiu et al., 2014; Rao et al., 2014), we next investigated whether FGF21 regulates the number or recruitment of these two types of type 2 immune cells in scWAT. To this end, SVF from scWAT was stained with antibodies specific for cell surface markers of eosinophils (F4/80, CD11b, and sialic acid-binding immunoglobulin receptor [Siglec F]) or ILC2s (CD45, CD25, and CD127), followed by flow cytometric analysis (Figures S4A and S4B). Cold exposure for 2 days induced an approximately 4-fold increase in the abundance of eosinophils in SVF obtained from scWAT in control mice (Figure 3E). However, this induction was significantly reduced by approximately 50% in A-FGF21KO mice (Figure 3E). In contrast, there was no obvious difference in the abundance of ILC2s in SVF of scWAT between A-FGF21KO mice and control littermates under both thermoneutral and cold conditions (Figure 3F). Consistently, adipose deficiency of FGF21 had no obvious impact on mRNA expression of IL-5 (Figure 3D), which is mainly derived from ILC2 cells (Lee et al., 2015; Molofsky et al., 2013). These findings collectively suggest that adipose-derived FGF21 is required for recruitment of eosinophils into scWAT during cold challenge. Cold-Induced Proliferation and Commitment of Adipocyte Precursor Cells and Accumulation of M2 Macrophages Is Compromised in scWAT of A-FGF21KO Mice APs, which are defined as Lin
CD31– CD45– Sca-1+ PDGFRa+ cells, are bipotential precursor cells that are able to be differentiated into both white and beige adipocytes (Lee et al., 2012). Notably, eosinophil-secreted type 2 cytokine (IL-4) promotes the proliferation and commitment of APs and their subsequent differentiation into beige adipocytes, thereby mediating beiging and thermogenesis of scWAT during cold challenge (Uhm and Saltiel, 2015). Therefore, we next investigated the impact of depletion of adipose FGF21 on the proliferating capacity of APs in SVF from scWAT with flow cytometric analysis (Figure S4C). Cold exposure for 2 days led to a marked elevation in the percentage of proliferating APs in scWAT of control mice (as assessed by nuclear staining for the cell proliferation marker Ki67) (Figure 3G). However, such cold exposure-mediated

induction in proliferation of APs was largely abrogated in A-FGF21KO mice (Figure 3G). Flow cytometric analysis for the beige lineage markers TMEM26 and CD137 (Figure S4C) showed that cold exposure for 2 days significantly increased abundance of committed beige-specific APs in SVF from scWAT, but such cold-induced commitment was largely compromised in A-FGF21KO mice (Figure 3H). Consistently, real-time PCR analysis showed that cold-induced upregulation of genes for markers of APs (Pdgfra and IL-4Ra) and markers of committed beige-specific APs (Tmem26, Tnfrsf9, also known as Cd137, and Ear2) in scWAT was significantly reduced in A-FGF21KO mice compared with the control littermates (Figure 3I). Apart from APs, M2 macrophages are another type of downstream effectors of eosinophils obligatory for cold-induced beiging of scWAT by producing catecholamines in the local adipose tissue (Qiu et al., 2014; Rao et al., 2014). Therefore, we also examined the roles of adipose-derived FGF21 on abundance of M2 macrophages (defined as F4/80hi CD11b+ CD206+ cells) in SVF from scWAT (Figure S4D). In the control mice, cold exposure for 2 days increased the frequency of M2 macrophages in scWAT by approximately 1.5-fold relative to the thermoneutral controls (Figure 3J). However, cold-induced increase in M2 macrophages was largely abrogated in A-FGF21KO mice (Figure 3J). On the other hand, the number of M1 macrophages (defined as F4/80hi CD11b+ CD11c+ cells) in SVF from scWAT was comparable between A-FGF21KO mice and wild-type control littermates under both thermoneutral and cold temperatures (Figure 3J). Cold Exposure Induces the Production of CCL11 in scWAT via Autocrine Actions of FGF21 in Adipocytes As eosinophils are recruited to target tissues mainly by the eotaxin subfamily of chemokines, including C-C motif chemokine ligands (CCLs) 11, 24, and 26 (also known as eotaxin-1, eotaxin-2, and eotaxin-3, respectively) via their surface receptor chemokine C-C motif receptor 3 (Jose et al., 1994), and acute administration of rmFGF21 has been shown to upregulate CCL11 expression in inguinal white adipose tissue (iWAT) in C57BL/6 mice (Muise et al., 2013), we thus investigated whether cold-induced FGF21 promotes recruitment of eosinophils into scWAT via induction of these chemokines.

Figure 3. Adipose-Derived FGF21 Is Required for Cold-Induced Recruitment of Eosinophils and M2 Macrophages, and Proliferation and Commitment of Adipocyte Precursor Cells in scWAT (A and B) SVF isolated from scWAT of 12-week-old male C57BL/6J mice was differentiated into primary adipocytes for 8 days and treated with recombinant FGF21 (rmFGF21, 1 mg/mL) or PBS (as a vehicle control) for 48 hr. The mRNA expression of Ucp1 (A) and the UCP1 protein expression (B) in the adipocytes were measured by real-time PCR and western blot analyses, respectively. Rosiglitazone (Rosi) (1 mM, dissolved in DMSO) was used as a positive control for induction of UCP1. (C–J) Inguinal scWAT was harvested from 12-week-old male A-FGF21KO mice and their control littermates housed at 30 C for 2 weeks before subjecting to exposure at 6 C or 30 C for 2 days. The mRNA expression of Adiponectin (Adn) (C), IL-4, IL-13, and IL-5 (D) in scWAT as determined by real-time PCR analysis. (E) Representative fluorescence-activated cell sorting (FACS) plots showing the frequency of eosinophils (F4/80+ CD11b+ Siglec F+ SSChi) in SVF. The right panel is the quantification of the percentage of eosinophils in total SVF cells from scWAT. (F) Representative FACS plots showing the abundance of type 2 innate lymphoid cells (ILC2s, Lin
CD5– CD45+ CD25+ CD127+) in SVF. The right panel is the quantification of the percentage of ILC2s in Lin
cells. (G) Representative FACS plots showing the proliferating adipocyte precursors (APs) (Lin
CD31– CD45– Sca-1+ and PDGFRa+) in SVF as determined by staining for Ki67 in APs. The right panel is the quantification of the percentage of proliferating cells in APs. (H) Expression of the beige-specific markers, TMEM26 and CD137, on APs. The upper panels are representative histograms of FACS analysis for TMEM26 and CD137. The lower panels are quantifications of mean fluorescence intensity (MFI) of TMEM26 and CD137 in APs. (I) The mRNA expression of the AP markers (Pdgfra and IL-4Ra) and beige lineage-specific markers (Tmem26, Tnfrsf9, and Ear2) in scWAT as determined by real-time PCR analysis. (J) Representative FACS plots of macrophages (M1, F4/80hi CD11b+ CD11c+; M2, F4/80hi CD11b+ CD206+) in SVF. Right panels are quantifications for the percentage of M1 and M2 macrophages in total macrophages (M4). Data represent mean ± SEM; n = 5–6 per group; repeated with three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S4.

Cell Metabolism 26, 1–16, September 5, 2017 7

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Figure 4. FGF21 Mediates Cold-Induced Expression of CCL11 via ERK1/2 in White Adipocytes

(A) The mRNA expression of Ccl11, Ccl24, and Ccl26 in scWAT of 12-week-old male C57BL/6J mice subjected to 6 C exposure for various time periods was measured by real-time PCR analysis. (B and C) Twelve-week-old A-FGF21KO mice or their control littermates were housed at 30 C for 2 weeks before exposure at 6 C or 30 C for another 2 days. The mRNA expression of Ccl11 in scWAT (B) and serum level of CCL11 (C) determined by real-time PCR analysis and ELISA, respectively. (D) The mRNA expression of Ccl11 in mature adipocytes (MA) and SVF fractionated from scWAT of 12-week-old male C57BL/6J mice subjected to 6 C or 30 C for 2 days was determined by real-time PCR analysis. (E and F) SVF cells isolated from scWAT of C57BL/6J mice were differentiated to white adipocytes, followed by treatment with rmFGF21 (1 mg/mL) or PBS (as a vehicle control) for 24 hr. The mRNA expression of Ccl11 in differentiated adipocytes (E) and CCL11 protein secreted into conditioned medium (F) was measured by real-time PCR analysis and ELISA, respectively. (G) Primary adipocytes were pre-incubated with the ERK inhibitors PD98059 (30 mM) and U0126 (10 mM), phosphatidylinositide 3-kinase inhibitor LY294002 (50 mM), or DMSO as control for 1 hr before treatment with rmFGF21 (1 mg/mL) for 24 hr. The mRNA expression of Ccl11 was determined by real-time PCR analysis. Data represent mean ± SEM; n = 4–6 per group; repeated with three independent experiments; **p < 0.01, ***p < 0.001; n.s., not statistically significant.

To this end, we tested whether the expression of CCL11 in scWAT is induced by cold challenge. Real-time PCR analysis demonstrated a progressive increase in mRNA expression of Ccl11 in scWAT from 6 hr to 3 days after cold challenge (Figure 4A). Notably, cold-induced expression of Ccl11 lagged slightly behind the induction of Fgf21 transcription in this tissue (Figure 1A). On the other hand, the expression levels of other two eotaxins, Ccl24 and Ccl26, were not altered during cold exposure (Figure 4A). Intriguingly, cold-induced expression of Ccl11 was markedly reduced in scWAT of A-FGF21KO mice by approximately 60% (p = 0.0011) (Figure 4B). Such a change was accompanied by a significant reduction in the circulating level of CCL11 in A-FGF21KO mice after cold exposure (Figure 4C). Although CCL11 was originally identified in endothelial cells and monocytes (Rothenberg et al., 1995), its expression has 8 Cell Metabolism 26, 1–16, September 5, 2017

also been detected in 3T3-L1 adipocytes in a differentiationdependent manner (Kim et al., 2011). Therefore, we next investigated which type of cells in scWAT is the main source for cold-induced production of CCL11. Real-time PCR analysis demonstrated that the expression level of Ccl11 was comparable between mature adipocytes and SVF isolated from scWAT of mice housed under the thermoneutral condition (Figure 4D). Interestingly, cold exposure for 2 days caused a greater than 4-fold increase in mRNA expression of Ccl11 in mature adipocytes, whereas there was no obvious elevation of Ccl11 expression in SVF after cold exposure (Figure 4D), indicating that cold challenge-induced CCL11 is predominantly produced from mature adipocytes, but not from other types of cells in SVF. To further investigate whether FGF21 directly acts on mature adipocytes to induce production of CCL11, SVF from scWAT

Please cite this article in press as: Huang et al., The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity, Cell Metabolism (2017), http://dx.doi.org/10.1016/j.cmet.2017.08.003

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Figure 5. Ablation of b-Klotho in Mature Adipocytes Impairs Cold-Induced Recruitment of Eosinophils and Biogenesis of Beige Cells in scWAT Twelve-week-old male A-KLBKO mice and their KLBlox/lox (control) littermates housed at 30 C for 2 weeks were subjected to cold exposure at 6 C or continued to be housed at 30 C for the indicated time periods. (A) Rectal temperature of mice housed at 30 C or 6 C for 24 hr. (B–H) scWAT collected from mice housed at 6 C or 30 C for 2 days were subjected to analysis for the mRNA expression of Ccl11 in scWAT by real-time PCR (B), the percentage of eosinophils in SVF (C), the percentage of proliferating APs (D), the MFI of beige-specific adipocyte precursor cell markers TMEM26 (E) and CD137 (F), and the percentages of M1 (G) and M2 (H) macrophages in total macrophages from scWAT were determined by flow cytometric analysis as in Figure 3. (legend continued on next page)

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of C57BL/6J mice was differentiated into adipocytes, followed by treatment with rmFGF21. Both real-time PCR analysis and ELISA demonstrated that treatment with rmFGF21 led to a robust induction in the mRNA expression (Figure 4E) and secretion (Figure 4F) of CCL11 in mature adipocytes. Notably, FGF21-induced expression of Ccl11 was blocked by PD98059 and U0126 (selective inhibitors of ERK), but not by the phosphatidylinositide 3-kinase inhibitor LY294002 (Figure 4G), suggesting that induction of Ccl11 by FGF21 is mediated by the canonical ERK1/2 signaling pathway. These findings, together with the fact that cold-mediated induction of Fgf21 is derived predominantly from mature adipocytes (Figure 1I), support the notion that induction of CCL11 in scWAT by cold challenge is attributed to autocrine actions of FGF21 in mature adipocytes. KLB in Mature Adipocytes Is Indispensable for Cold-Induced Recruitment of Eosinophils and Beige Fat Thermogenesis FGF21 exerts its metabolic regulation in adipocytes by binding to its receptor FGFR1 and co-receptor KLB, the latter of which is a single transmembrane glycoprotein that determines the tissue specificity of FGF21 actions (Fisher and Maratos-Flier, 2016). To interrogate whether the actions of FGF21 in adipocytes are obligatory for cold-induced recruitment of eosinophils and ensuing beiging in scWAT, we generated adipocyte-specific KLB KO (A-KLBKO) mice by crossing Klb gene floxed mice with mice overexpressing Cre driven by the promoter of the Adiponectin gene (Lin et al., 2015) (Figure S5A). Notably, deletion of KLB in adipocytes did not affect the production of Fgf21 in either scWAT (Figure S5B) or BAT (Figure S5C) in mice housed under thermoneutral or cold conditions. Similar to A-FGF21KO mice, A-KLBKO mice exhibited significantly lower rectal temperature than that in the KLBlox/lox (control) mice after cold exposure for 6 days (Figure 5A). The cold-induced mRNA expression of Ccl11 in scWAT of A-KLBKO mice was largely compromised compared with their control littermates (Figure 5B). Such a change was accompanied by a significant reduction in coldinduced recruitment of eosinophils into scWAT in A-KLBKO mice (Figure 5C). In addition, adipocyte-specific deficiency of KLB significantly reduced cold-induced proliferation and commitment of APs in scWAT, as demonstrated by downregulation of Ki67 (Figure 5D), TMEM26 (Figure 5E) and CD137 (Figure 5F) in flow cytometric analyses. These changes in scWAT of A-KLBKO mice were also paralleled by a marked reduction in cold-induced accumulation of the adipose-resident M2 macrophages (Figure 5H). Consequently, cold-induced expression of thermogenic genes (Figure 5I) and UCP1 protein expression (Figure 5J) in scWAT were markedly reduced in A-KLBKO mice compared with the controls. Histological analysis further confirmed that there were significantly fewer multilocular (Figure 5K) and UCP1+ (Figure 5L) beige adipocytes in scWAT of A-KLBKO

mice than that of control mice after cold exposure for 6 days. On the other hand, deletion of KLB in adipocytes did not affect thermogenic gene expression (Figure S5D), adipocyte morphology (Figure S5F) or UCP1 protein levels (Figures S5E and S5G) in BAT at both thermoneutral and cold temperatures. Taken together, these findings demonstrate that autocrine actions of FGF21 on mature adipocytes through KLB are required for expression of CCL11, recruitment of eosinophils and beiging in scWAT during cold adaptation. CCL11 Is a Physiological Mediator for Cold-Induced Recruitment of Eosinophils and Beiging in scWAT To address whether CCL11 is a physiological regulator of type 2 immune responses and beiging in scWAT, C57BL/6J mice were injected subcutaneously with a rat anti-mouse CCL11 neutralizing antibody to block the actions of endogenous CCL11 (Villeda et al., 2011), followed by exposure at 6 C or 30 C for 2 or 3 days (Figure 6A). Cold exposure-induced elevations in both abundance of eosinophils (Figure 6B) and mRNA expression of eosinophil markers (Siglec F and IL-4) (Figure 6C) in scWAT were markedly reduced by the neutralizing antibody to a basal level under the thermoneutral condition, whereas treatment with an isotype antibody (rat nonimmune immunoglobulin G, as a control) did not have such an effect, suggesting that CCL11 is a physiological chemoattractant required for accumulation of eosinophils in scWAT during adaptation to cold exposure. Consistently, cold exposure-induced expressions of the bipotential AP marker (Pdgfra), beige lineage-specific markers (Tmem26 and Tnfrsf9) (Figure 6D), and thermogenic genes (Figure 6E) in scWAT of mice were all significantly suppressed by treatment with the neutralizing antibody against CCL11. Such changes were paralleled by markedly impaired induction of UCP1 protein (Figure 6F) and reduced morphological conversion from white to beige adipocytes (Figures 6G and 6H) in scWAT of anti-CCL11-treated mice after cold challenge for 3 days. CCL11 Is Obligatory for FGF21-Mediated Effects on Beiging of scWAT during Cold Adaptation Given that cold-induced production of CCL11 in scWAT is attributed to the autocrine actions of FGF21, we next investigated whether the stimulatory effects of FGF21 on beiging of scWAT is dependent on CCL11. To this end, A-FGF21KO mice housed at 30 C were pre-treated with an anti-CCL11 neutralizing antibody or isotype control 1 day prior to local injection of rmFGF21 protein into subcutaneous fat pad, followed by exposing the mice to 6 C with daily injection of both anti-CCL11 and/or rmFGF21 protein (Figure S6A). In A-FGF21KO mice treated with the isotype control, replenishment with rmFGF21 reversed the impairments in cold-induced recruitment of eosinophils (Figures S6B and S6C) and beiging remodeling of scWAT (Figures S6D–S6I) to a level comparable with that in control mice

(I and J) The mRNA expression of several thermogenic genes (I) and protein expression of UCP1 (J) in scWAT from mice housed at 6 C or 30 C for 6 days as determined by real-time PCR and western blot analyses, respectively. The right panel of (J) is the densitometric analysis for the relative abundance of UCP1 normalized with b-Tubulin. (K and L) Representative images of H&E staining (K) and immunohistochemical staining for UCP1 (L) in scWAT from mice exposed to 6 C or 30 C for 6 days. Scale bar, 20 mm. Data represent mean ± SEM; n = 5–6 per group; repeated with three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5.

10 Cell Metabolism 26, 1–16, September 5, 2017

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Figure 6. CCL11 Is Obligatory for Cold-Induced Recruitment of Eosinophils and Beiging in scWAT (A) Schematic diagram for the treatment regime. Twelve-week-old male C57BL/6J mice housed at 30 C for 2 weeks were treated with a neutralizing antibody against CCL11 (anti-CCL11, 100 mg/kg/day) or the same amount of isotype antibody by daily bilateral subcutaneous injection into inguinal scWAT and then housed at 6 C or 30 C. (B–D) scWAT was harvested at 2 days after exposure at 6 C or 30 C and analyzed for the percentage of eosinophils in SVF by flow cytometry (B), and mRNA expression of eosinophil markers (Siglec F and IL-4) (C) and beige-specific AP markers (D) by real-time PCR analysis. (E and F) scWAT collected at 3 days after exposure at 6 C or 30 C were analyzed for the mRNA expression of a panel of thermogenic genes by real-time PCR analysis (E), and protein expression of UCP1 by western blot analysis (F). The right panel of (F) is the densitometric analysis for the relative abundance of UCP1 normalized with b-Tubulin. (G and H) Representative images of H&E staining (G) and immunohistochemistry for UCP1 (H) in scWAT from mice exposed to 6 C or 30 C for 3 days. Scale bar, 20 mm. Data represent mean ± SEM; n = 5–6 per group; repeated with three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001.

Cell Metabolism 26, 1–16, September 5, 2017 11

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Figure 7. Replenishment of Recombinant CCL11 Reverses Impairments in Cold-Induced Beiging of scWAT in A-FGF21KO Mice (A) Schematic diagram for the experimental procedures. Twelve-week-old male A-FGF21KO mice housed at 30 C for 2 weeks were treated with recombinant mouse CCL11 protein (20 mg/kg/day) or PBS by daily bilateral subcutaneous injection into inguinal scWAT and subjected to cold challenge (6 C). Wild-type control mice receiving PBS were used as a control. (B–F) scWAT harvested at 2 days after cold challenge or thermoneutral exposure was analyzed for the percentage of eosinophils in SVF by flow cytometry (B) and mRNA expression of eosinophil markers (Siglec F and IL-4) (C) and beige-specific adipocyte precursor markers (D) by real-time PCR analysis. The mRNA expression level of several thermogenic genes (E) and protein expression of UCP1 (F) in scWAT collected at 3 days post cold challenge or thermoneutral exposure were measured by real-time PCR and western blot analysis, respectively. The right panel of (F) is the densitometric analysis for the relative abundance of UCP1 normalized with b-Tubulin. (legend continued on next page)

12 Cell Metabolism 26, 1–16, September 5, 2017

Please cite this article in press as: Huang et al., The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity, Cell Metabolism (2017), http://dx.doi.org/10.1016/j.cmet.2017.08.003

exposed to 6 C (Figures 6B–6H). However, these effects induced by local injection of rmFGF21 were largely abrogated in A-FGF21KO mice pre-treated with the anti-CCL11 neutralizing antibody, with respect to recruitment of eosinophils (Figures S6B and S6C), commitment of APs (Figure S6D), accumulation of M2 macrophages (Figure S6E), induction of thermogenic genes and proteins (Figures S6F and S6G), and formation of beige adipocytes (Figures S6H and S6I). As cold-induced expression of CCL11 in scWAT is abolished in A-FGF21KO mice, we next examined whether supplementation of recombinant CCL11 protein could rescue the defects in beiging of scWAT in A-FGF21KO mice after cold exposure. Treatment of A-FGF21KO mice with recombinant mouse CCL11 protein (Figure 7A) led to a significant restoration of cold-induced accumulation of eosinophils in scWAT to a level comparable with that in control mice exposed to a cold environment, as determined by both flow cytometric (Figure 7B) and real-time PCR (Figure 7C) analyses. These changes caused by replenishment of recombinant CCL11 were accompanied by markedly increased expression of the committed beige AP markers (Figure 7D) and thermogenic genes (Figure 7E), as well as a significant induction of UCP1 protein expression (Figure 7F) in scWAT of A-FGF21KO mice compared with vehicle-treated A-FGF21KO mice. Likewise, CCL11-treated A-FGF21KO mice also exhibited a significant elevation of coldinduced formation of multilocular beige-like cells (Figure 7G) and an increase in the number of UCP1+ adipocytes (Figure 7H) in scWAT to a level equivalent to the controls exposed to cold environment. Taken together, these observations demonstrate that supplementation of CCL11 is sufficient to reverse the impairments in cold-induced recruitment of eosinophils into scWAT, restoring cold-induced beiging of scWAT in A-FGF21KO mice. DISCUSSION Despite extensive investigations on metabolic functions of FGF21 in recent years, its role in immune regulation has scarcely been explored. Our present study provides a series of evidence demonstrating that adipose-derived FGF21 is a key triggering factor for activation of type 2 immune responses via its autocrine actions to induce the recruitment of eosinophils, which is the crucial step for beiging and adaptive thermogenesis of scWAT in response to cold challenges. These findings support the role of FGF21 as a physiological integrator of metabolism and immunity, by mediating the crosstalk between mature adipocytes and immune cells in WAT. Type 2 immune cells residing in adipose tissues play a crucial role in maintaining energy homeostasis and insulin sensitivity. In particular, eosinophils are a major source of type 2 cytokines in healthy WAT, and its abundance in WAT is reduced in both dietary and genetically obese mice (Wu et al., 2011). Mice lacking eosinophils (such as the DdbjGATA mutant mice) are more susceptible to dietary obesity and insulin resistance with

reduced number of adipose-resident M2 macrophages than their wild-type littermates (Wu et al., 2011). In contrast, mice with transgenic overexpression of IL-5 or infection with helminth pathogens, both of which cause accumulation of eosinophils in adipose tissues, are protected from diet-induced obesity and insulin resistance (Wu et al., 2011; Yang et al., 2013). Deletion of eosinophils or blocking its type 2 cytokine signaling in mice reduces the development of beige adipocytes and causes hypothermia during cold exposure (Qiu et al., 2014). Previous studies have identified ILC2s as an upstream regulator for the accumulation of eosinophils in WAT through the production of IL-5 (Molofsky et al., 2013). However, ILC2s also enhance beige adiopogenesis independent of eosinophils, by producing methionine-enkephalin peptide to induce UCP1 expression in WAT (Brestoff et al., 2015). Cold challenge has no obvious effect on either the abundance of ILC2s or expression of IL-5 in WAT (Hui et al., 2015). The physiological stimulator(s) that trigger(s) the recruitment of eosinophils under cold conditions remain elusive. In this regard, our present study identifies adiposederived FGF21, which is induced by over 60-fold in response to cold challenge, as an obligatory endogenous mediator of cold-induced recruitment of eosinophils through its autocrine actions to induce CCL11 production in mature adipocytes. Mice with adipose tissue-selective depletion of FGF21 or its co-receptor KLB exhibit impairments in cold-induced recruitment of eosinophils into scWAT accompanied with reduced number of M2 macrophages and diminished biogenesis of beige cells, a phenotype strikingly resembling that in eosinophil-deficient mice (Qiu et al., 2014). CCL11 (also known as eosinophil chemotactic protein and eotaxin-1) is a member of the chemokine C-C motif ligand family that selectively recruits eosinophils by their chemotaxis, and is therefore a key player in allergic responses (Jose et al., 1994). Although the association between adipose CCL11 expression and obesity has been observed in both animals and human, and adipose-derived CCL11 has been suggested to be an important contributor to circulating CCL11 in obese individuals (Huber et al., 2008; Vasudevan et al., 2006), the physiological roles of CCL11 in metabolic regulation have never been explored. In the present study, we demonstrate that endogenous CCL11 is an obligatory downstream effector of FGF21 to mediate cold-induced recruitment of eosinophils in scWAT, which in turn induces the beiging and thermogenic program. The expression levels of both CCL11 and FGF21 are selectively induced in mature adipocytes (Figures 1I and 4D), whereas this cold-induced CCL11 is abolished in mice with adipose tissue-selective depletion of either FGF21 or its co-receptor KLB (Figures 4B, 4C, and 5B). In line with our findings, a previous microarray-based study also identified CCL11 in WAT as one of the downstream target proteins induced by pharmacological treatment with rmFGF21 in mice (Muise et al., 2013). Notably, both cold challenge and FGF21-induced recruitment of eosinophils (Figures 6B and S6B), accumulation of M2 macrophages (Figure S6E) and proliferation and commitment of

(G and H) Representative images of H&E staining (G) and immunohistochemistry for UCP1 (H) in scWAT from mice at 3 days post cold challenge or thermoneutral exposure. Scale bar, 20 mm. Data represent mean ± SEM; n = 5–6 per group; repeated with three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not statistically significant. See also Figure S6.

Cell Metabolism 26, 1–16, September 5, 2017 13

Please cite this article in press as: Huang et al., The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity, Cell Metabolism (2017), http://dx.doi.org/10.1016/j.cmet.2017.08.003

APs (Figures 6D and S6D) are abrogated by neutralization of CCL11 actions in scWAT, whereas replenishment with recombinant CCL11 is sufficient to reverse the impairments in coldinduced enrichment of eosinophils and biogenesis of beige cells in scWAT of A-FGF21KO mice (Figure 7). Taken together, the data from both gain-of-function and loss-of-function experiments support the role of adipose-derived FGF21-CCL11 axis as a physiological driver for type 2 immune activation and beiging of scWAT. In primary adipocytes, we found that FGF21 stimulates CCL11 production through the KLB-ERK1/2 signaling cascade. Consistently, ERK1/2 has been shown to mediate both IL-17A- and IL-9-induced CCL11 expression by phosphorylating the transcription factor STAT3 to transactivate the Ccl11 gene promoter in human airway smooth muscle cells (Saleh et al., 2009; Yamasaki et al., 2010). FGF21 induces ERK1/2 activation via the FGFR1/KLB receptor complex, which in turn mediates the metabolic actions of FGF21 on modulation of glucose uptake and lipolysis in adipocytes (Fisher and Maratos-Flier, 2016; Kharitonenkov et al., 2008). ERK1/2 has also been implicated in beiging and thermogenesis of WAT or activation of classical BAT induced by a number of thermogenic agents, including irisin and transient receptor potential vanilloid 4 (Ye et al., 2012; Zhang et al., 2014). Further investigations are needed to interrogate whether ERK1/2 activation in adipocytes is sufficient to induce beiging of WAT by driving CCL11-mediated recruitment of eosinophils. Expression of FGF21 in adipocytes and hepatocytes is differentially regulated, and exhibits opposite changes during the fasting-feeding cycle in mice (Dutchak et al., 2012; Inagaki et al., 2007). Notably, both cold exposure and b3-adrenergic stimulation strongly induce FGF21 expression in adipocytes (but not in the liver) by cyclic-AMP-mediated activation of protein kinase A and p38 MAPK, which in turn phosphorylates the transcription factor ATF2 for transactivation of the Fgf21 gene promoter (Fisher et al., 2012; Hondares et al., 2011), suggesting that adipose FGF21 is a downstream effector of sympathetic nerve activation. Therefore, despite the poor sympathetic innervation in WAT, the dramatic induction of FGF21 in adipocytes serves as a compensatory mechanism to amplify the signal of sympathetic tone by triggering type 2 immune responses in the local subcutaneous adipose tissue, thereby creating a microenvironment favorable for beiging and thermogenesis during cold exposure. While our present study shows adipose-derived FGF21 as a physiological mediator of cold-induced beiging via its adipocyte-autonomous actions, FGF21 at a pharmacological dose has been shown to act centrally to induce the activity of classical BAT (Owen et al., 2014). Mice with transgenic overexpression of Fgf21 or chronic administration of rmFGF21 exhibit increased sympathetic outflow to BAT through a neuropeptide corticotropin-releasing factor-dependent mechanism (Owen et al., 2014). On the other hand, either global or adipose tissue-selective knock out of FGF21 in mice only causes impairments in cold-induced beiging of scWAT, but has no effect on classical BAT activity (Fisher et al., 2012) (Figures 2 and S2). We did not detect any obvious difference in the hypothalamic expression of Crf or adipose expression of Th between A-FGF21KO mice and their control littermates housed at either thermoneutral or cold temperatures (our unpublished 14 Cell Metabolism 26, 1–16, September 5, 2017

data). This discrepancy can be reconciled by the discordance between pharmacological effects and physiological actions of FGF21, a phenomenon observed in many previous studies (Kliewer and Mangelsdorf, 2010). There is growing evidence suggesting that beige adipocytes, once activated, are metabolically functional, playing an active role in whole-body energy expenditure and thermogenesis in response to cold and other metabolic stresses. Mice with adipose overexpression of HOXC10 (a homeobox domain-containing transcription factor) exhibit selective impairment in beiging of scWAT with normal BAT activity, which is accompanied by lower body temperature and decreased cold-induced glucose clearance (Ng et al., 2017). Vice versa, mice with transgenic expression of miR-196a or depletion of gut microbiota, both of which enhance beiging of scWAT without obvious effects on BAT function, display increased energy expenditure and core body temperature, and are resistant to diet-induced obesity (Mori et al., 2012; Sua´rez-Zamorano et al., 2015). In human adults, constitutively active BAT is hardly detectable under ambient temperature, whereas cold exposure-induced active brown adipocytes resemble more to inducible beige adipocytes (Wu et al., 2012). Furthermore, in both rodents and humans, beige adipocytes have additional metabolic benefits beyond its effects on thermogenic activities, including improvement of systemic glucose homeostasis and insulin sensitivity, and reduction of cardiometabolic risk by increasing HDL turnover and increasing cholesterol turnover (Bartelt et al., 2017; Hanssen et al., 2015; Stanford et al., 2015). In this connection, FGF21-mediated beiging of scWAT may be physiologically important not only for whole-body thermogenesis induced by metabolic stresses such as cold exposure, but also for maintenance of systemic glucose and lipid metabolism as well as insulin sensitivity. In summary, our present study uncovers a novel physiological role of the adipose-derived FGF21-CCL11 axis in coupling sympathetic nerve activation with type 2 immune responses in scWAT to coordinate beiging and adaptive thermogenesis during cold exposure, and also suggests that cold-induced recruitment of eosinophils and type 2 immune activation in scWAT are driven by the signals originated from mature adipocytes. Although both animal and clinical studies have shown the promising effects of FGF21 in reducing body weight and fat mass, the therapeutic benefits of recombinant FGF21 in obese individuals may be compromised by the existence of FGF21 resistance (Chui et al., 2010). Indeed, reduced expression of the FGF21 receptor complex in adipose tissues has been observed in both rodents and humans with obesity (Chui et al., 2010; Dı´az-Delfı´n et al., 2012). In this connection, our present study raises the possibility that pharmacological interventions targeting adipose CCL11, which is a downstream effector of KLB in the FGF21 signaling cascades, may represent an alternative therapeutic strategy for obesity with FGF21 resistance. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING

Please cite this article in press as: Huang et al., The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity, Cell Metabolism (2017), http://dx.doi.org/10.1016/j.cmet.2017.08.003

d

d

d

EXPERIMENTAL MODEL AND SUBJECT DETAILS B Mice B In Vivo Treatments METHOD DETAILS B Indirect Calorimetry B Temperature Measurements B Ex Vivo Oxygen Consumption in Adipose Tissues B Flow Cytometry B In Vitro Experiments in Primary Adipocytes B Western blot, ELISA, and Histological Analyses B RNA Extraction and Real-Time PCR QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and one table and can be found with this article online at http://dx.doi.org/10.1016/j.cmet.2017.08.003. AUTHOR CONTRIBUTIONS Z.H. designed the study, carried out the research, analyzed and interpreted the results, and wrote the manuscript. L.Z. and J.Z. provided reagents and technical assistance for the main experiments. J.T.H.L. assisted with main experiments. D.W. and L.G. provided essential mouse lines for this study. Y.W. reviewed the manuscript. C.M.W. discussed the results and wrote and reviewed the manuscript. A.X. conceived and supervised the study and wrote and edited the manuscript. ACKNOWLEDGMENTS This work was supported by National 973 Basic Research Program of China (2015CB553603), Research Grant Council of Hong Kong (17161016 and C7055-14G), National Health and Medical Research Council of Australia (NHMRC)/National Science Foundation of China (NSFC) Joint Research Program (81561128016), and HKU matching fund for the State Key Laboratory of Pharmaceutical Biotechnology. Received: February 26, 2017 Revised: May 24, 2017 Accepted: August 1, 2017 Published: August 24, 2017 REFERENCES Adams, A.C., Coskun, T., Cheng, C.C., Libbey, S., DuBois, S.L., and Kharitonenkov, A. (2013). Fibroblast growth factor 21 is not required for the antidiabetic actions of the thiazoladinediones. Mol. Metab. 2, 205–214. Bartelt, A., John, C., Schaltenberg, N., Berbe´e, J.F., Worthmann, A., Cherradi, M.L., Schlein, C., Piepenburg, J., Boon, M.R., and Rinninger, F. (2017). Thermogenic adipocytes promote HDL turnover and reverse cholesterol transport. Nat. Commun. 8, 15010. Brestoff, J.R., Kim, B.S., Saenz, S.A., Stine, R.R., Monticelli, L.A., Sonnenberg, G.F., Thome, J.J., Farber, D.L., Lutfy, K., Seale, P., et al. (2015). Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity. Nature 519, 242–246. Chang, L., Villacorta, L., Li, R., Hamblin, M., Xu, W., Dou, C., Zhang, J., Wu, J., Zeng, R., and Chen, Y.E. (2012). Loss of perivascular adipose tissue upon PPARg deletion in smooth muscle cells impairs intravascular thermoregulation and enhances atherosclerosis. Circulation 126, 1067–1078. Chau, M.D., Gao, J., Yang, Q., Wu, Z., and Gromada, J. (2010). Fibroblast growth factor 21 regulates energy metabolism by activating the AMPK– SIRT1–PGC-1a pathway. Proc. Natl. Acad. Sci. USA 107, 12553–12558. Chui, P.C., Antonellis, P.J., Bina, H.A., Kharitonenkov, A., Flier, J.S., and Maratos-Flier, E. (2010). Obesity is a fibroblast growth factor 21 (FGF21)-resistant state. Diabetes 59, 2781–2789.

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Please cite this article in press as: Huang et al., The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity, Cell Metabolism (2017), http://dx.doi.org/10.1016/j.cmet.2017.08.003

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Rat monoclonal anti-mouse CCL11 (clone 42285)

R&D

Cat#MAB420-100; RRID: AB_2070629

Rat monoclonal IgG2A isotype control (clone 54447)

R&D

Cat#MAB006; RRID: AB_357349

Rat monoclonal anti-mouse F4/80-PE (clone CI:A3-1)

Abcam

Cat#ab105156; RRID: AB_10712176

Rat anti-mouse CD11b-FITC (clone M1/70)

BioLegend

Cat#101205; RRID: AB_312788

Rat anti-mouse CD11b-BV421 (clone M1/70)

BD Biosciences

Cat#562605; RRID: AB_11152949

Rat monoclonal anti-mouse Siglec F-Alexa Fluor 647 (clone E50-2440)

BD Biosciences

Cat#562680

Lineage cocktail-FITC (clone 145-2C11, RB6-8C5, M1/70, RA3-6B2, Ter-119)

BioLegend

Cat#78022

Rat anti-mouse CD31-FITC (clone 390)

BioLegend

Cat#102405; RRID: AB_312900

Rat anti-mouse CD45-FITC (clone 30-F11)

BioLegend

Cat#103107; RRID: AB_312972

Rat anti-mouse PDGFRa (CD140a)-PE (clone APA5)

BioLegend

Cat#135905; RRID: AB_1953268

Rat anti-Sca1-Pacific Blue (clone D7)

BioLegend

Cat#108120; RRID: AB_493273

Rat monoclonal anti-mouse Ki67-APC (clone 16A8)

BioLegend

Cat#652405; RRID: AB_2561929

Syrian hamster anti-mouse CD137-APC (clone 17B5)

BioLegend

Cat#106109; RRID: AB_2564296

Rabbit anti-mouse TMEM26

Novus Biologicals

Cat#NBP2-27334SS

Goat anti-rabbit-PE-Cy7

Santa Cruz

Cat#sc-3845; RRID: AB_649109

Hamster monoclonal anti-mouse CD11c-FITC (clone HL3)

BD Biosciences

Cat#557400; RRID: AB_396683

Rat anti-mouse CD206-Alexa Fluor 647 (clone C068C2)

BioLegend

Cat#141712; RRID: AB_10900420

Rat anti-mouse CD45-Pacific Blue (clone 30-F11)

BioLegend

Cat#103125; RRID: AB_493536

Rat monoclonal anti-mouse CD25-PE (clone 3C7)

BD Biosciences

Cat#553075; RRID: AB_394605

Rabbit monoclonal anti-mouse IL-7Ra/CD127 (clone 1140A)

R&D

Cat#MAB7473-SP

Antibodies

Rat anti-mouse CD5-FITC (clone 53-7.3)

BioLegend

Cat#100605; RRID: AB_312734

Rabbit anti-mouse UCP1

Abcam

Cat#ab10983; RRID: AB_2241462

Rabbit anti-b-Tubulin

Cell Signaling Technology

Cat#2146; RRID: AB_2210545

Mouse monoclonal total OXPHOS rodent antibody cocktail

Abcam

Cat#ab110413; RRID: AB_2629281

Chemicals, Peptides, and Recombinant Proteins Endotoxin-free recombinant mouse FGF21 protein

Immunodiagnostics Limited

Cat#42189

Recombinant mouse CCL11 protein

R&D

Cat#420-ME-020/CF

Norepinephrine

Sigma-Aldrich

Cat#74480

Collagenase, Type I

Life Technologies

Cat#17100-017

Red blood cell (RBC) lysis buffer

BioLegend

Cat#420301

LIVE/DEAD fixable near-IR dead cell stain

Molecular Probes

Cat#L34975

Fixation/Permeabilization kit

eBioscience

Cat#00-5523

Dexamethasone

Sigma-Aldrich

Cat#D8893

3-isobutyl-1-methylxanthine (IBMX)

Sigma-Aldrich

Cat#I5879

Rosiglitazone

Sigma-Aldrich

Cat#R2408

Insulin

Novo Nordisk

Actrapid HM

Oil red O

Sigma-Aldrich

Cat#o9755

Protease inhibitor cocktail tablets

Roche

Cat#04 693 116 001

PD98059

Cell Signaling Technology

Cat#9900 (Continued on next page)

Cell Metabolism 26, 1–16.e1–e4, September 5, 2017 e1

Please cite this article in press as: Huang et al., The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity, Cell Metabolism (2017), http://dx.doi.org/10.1016/j.cmet.2017.08.003

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

U0126

Cell Signaling Technology

Cat#9903

LY294002

Cell Signaling Technology

Cat#9901

Enhanced chemiluminescence reagents

GE Healthcare

Cat#RPN2232

RNAiso Plus

Takara

Cat#9109

PrimeScript RT reagent kit

Takara

Cat#RR037A

SYBR Premix Ex Taq

Takara

Cat#RR420D

Mouse CCL11/Eotaxin Quantikine ELISA kit

R&D

MME00

Mouse FGF-21 Immunoassay Kit

Immunodiagnostics Limited

Cat#32180

DNeasy 96 Blood & Tissue Kit

Qiagen

Cat#69581

C57BL/6J

The Jackson Laboratory

Stock No: 000664

B6.Cg-Tg (Alb-cre) 21 Mgn/J (Albumin-Cre)

The Jackson Laboratory

Stock No: 003574

B6.Cg-Tg (Fabp4-cre) 1Rev/J (aP2-Cre)

The Jackson Laboratory

Stock No: 005069

Critical Commercial Assays

Experimental Models: Organisms/Strains

B6;FVB-Tg (Adipoq-cre) 1Evdr/J (adiponectin-Cre)

The Jackson Laboratory

Stock No: 010803

FGF21-floxed (FGF21 lox/lox)

Shanghai Nanfang Centre for Model Organisms

N/A

b-Klotho-floxed (KLB lox/lox)

Lin et al., 2015

N/A

Oligonucleotides Real-time PCR primers used in this study

See Table S1

N/A

Primers used for genotyping in this study

See Table S1

N/A

FlowJo version X.0.7

Tree Star

https://www.flowjo.com/solutions/flowjo

Image J

National Institutes of Health (NIH)

https://imagej.nih.gov/ij/

GraphPad Prism 7

GraphPad

https://www.graphpad.com/

FLIR Tools

FLIR

https://www.flir.com/

Falcon cell strainer, 70 mm

BD Biosciences

Cat#352350

Seahorse XFe24 Islet Capture FluxPak

Agilent Technologies

Cat#103518-100

Standard chow diet

LabDiet

Cat#5053

Software and Algorithms

Other

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents may be obtained from the Lead Contact Aimin Xu (The University of Hong Kong; Email: [email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Mice FGF21-floxed (FGF21lox/lox) and b-Klotho-floxed (KLBlox/lox) mice, the latter of which is described as previously (Lin et al., 2015), were generated by Shanghai Nanfang Centre for Model Organisms, and both were backcrossed with C57BL/6J mice for at least eight generations. Albumin-Cre, aP2-Cre, and adiponectin-Cre mice were obtained from the Jackson Laboratory. All these mice were on a C57BL/6J background. Adipose-selective FGF21 knockout (A-FGF21KO) and liver-selective FGF21 knockout (L-FGF21KO) mouse strains were generated by crossing the FGF21-floxed mice with aP2-Cre and albumin-Cre mice, respectively. Adipocyteselective b-Klotho knockout (A-KLBKO) mice were generated by crossing the b-Klotho-floxed mice with adiponectin-Cre mice. Mice were housed in a controlled environment (12-hour light-dark cycle, 23 C ± 1 C) with ad libitum access to water and standard chow diet (LabDiet 5053, LabDiet). For cold challenge experiments, 12-week-old male mice were maintained at the thermoneutral temperature (30 C) in the intensive care unit (ICU) for 2 weeks prior to cold exposure at 6 C in pre-chilled cages for various time periods in groups of two mice per cage. Mice housed at 30 C for the same period were used as thermoneutral controls. All animal studies were approved by Committee on the Use of Live Animals in Teaching and Research at the University of Hong Kong.

e2 Cell Metabolism 26, 1–16.e1–e4, September 5, 2017

Please cite this article in press as: Huang et al., The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity, Cell Metabolism (2017), http://dx.doi.org/10.1016/j.cmet.2017.08.003

In Vivo Treatments Endotoxin-free rmFGF21 protein was prepared as described previously (Lin et al., 2013). 12.5 mg rmFGF21 or 0.5 mg recombinant mouse CCL11 protein (R&D) dissolved in 50 ml phosphate-buffered saline (PBS) was directly administered into scWAT of A-FGF21KO mice daily by bilateral subcutaneous (s.c.) injection. For neutralization of adipocyte-secreted CCL11, 2.5 mg monoclonal rat antimouse CCL11 antibody (clone 42285, R&D) or monoclonal rat IgG2A isotype control antibody (clone 54447, R&D) dissolved in 50 ml PBS was directly administered into scWAT of A-FGF21KO and wild-type control mice daily by bilateral subcutaneous injection (Villeda et al., 2011). METHOD DETAILS Indirect Calorimetry Whole-body oxygen consumption (VO2) was measured using the comprehensive laboratory animal monitoring system (CLAMS, Columbus Instruments) as previously described (Hui et al., 2015). Briefly, mice were housed individually in CLAMS cages and acclimatized for 24 hours. VO2 was recorded every 11 min for a period of 48 hours at 30 C and 6 C. Data were calculated by normalizing with body weights. Temperature Measurements The body temperature was measured using a rectal probe connected to a digital thermometer (Powertronics). Surface temperature surrounding inguinal scWAT and interscapular BAT was recorded with a FLIR T440 infrared camera, and temperature was quantified by using FLIR analysis software. Ex Vivo Oxygen Consumption in Adipose Tissues Ex vivo oxygen consumption in adipose tissues was measured using an Seahorse XFe24 extracellular flux analyzer (Agilent Technologies) as previously described (Hui et al., 2015). Briefly, scWAT or BAT collected from 12-week-old male mice housed at 30 C and 6 C for 6 days was cut into pieces of approximately 2 mm3, weighed and equilibrated in Dulbecco’s Modified Eagle Medium (DMEM) without NaHCO3 (Thermo Fisher Scientific) at 37 C for 1 hour before measurement. Norepinephrine (NE) (10 mM, Sigma) was added sequentially during the assay to measure the basal and b3-adrenergic receptor-dependent oxygen consumption rate (OCR), respectively. The values were normalized with tissue weight. Flow Cytometry SVF was isolated from scWAT of mice as previously described (Hui et al., 2015). Briefly, the fat pads were minced with scissors and digested in 2 mg/ml collagenase (Life technologies) for 30 min at 37 C. The homogenates were filtered through a 70 mm cell strainer (BD Biosciences), followed by centrifugation at 800 x g for 15 min at 4 C. The pellets were incubated in red blood cell lysis buffer (Biolegend) for 5 min on ice, centrifuged at 800 x g for 15 min at 4 C, and resuspended in PBS buffer. Cells were centrifuged at 800 x g for 15 min at 4 C, resuspended in 1 ml of Live/dead fixable dead cell stain (Molecular Probes) and incubated on ice for 30 min. Afterwards, cells were washed once with FACS buffer (1% BSA in 1xPBS) followed by staining with different antibodies. For flow cytometric analysis of eosinophils, cells were triple-stained with antibodies: Rat monoclonal antibody (mAb) against mouse F4/80-Phycoerythrin (PE) (1:100, clone CI:A3-1, Abcam), Rat anti-mouse CD11b-BV421 (1:100, clone M1/70, BD Biosciences), and Rat mAb against mouse Siglec F-Alexa Fluor 647 (1:100, clone E50-2440, BD Biosciences) on ice for 30 min in dark. Species matched IgG (Biolegend) were used as non-specific isotype controls. For analysis of adipocyte precursors, cells were stained with lineage cocktail-FITC, which contains anti-CD3e, -Ly-6G, -Ly-6C, -CD11b, -CD45R/B220, -Ter-119 (1:200, clone 145-2C11, RB6-8C5, M1/70, RA3-6B2, Ter-119, Biolegend), to exclude other cell lineages from the analysis gates combined with antibodies: Rat anti-mouse CD31-FITC (1:100, clone 390, Biolegend), Rat anti-mouse CD45-FITC (1:100, clone 30-F11, Biolegend), Rat antimouse PDGFRa (CD140a)-PE (1:100, clone APA5, Biolegend), Rat anti-Sca1-Pacific BlueTM (1:100, clone D7, Biolegend), or their isotype controls (Biolegend) for 30 min on ice in dark. For analysis of Ki67 and beige markers (TMEM26 and CD137) in adipocyte precursors, cells were fixed and permeabilized using the Fixation/Permeabilization kit (eBioscience) as per manufacturer’s instructions before antibody staining with: Rat mAb against mouse Ki67-Allophycocyanin (APC) (1:80, clone 16A8, Biolegend) or Syrian hamster anti-mouse CD137-APC (1:100, clone 17B5, Biolegend), Rabbit anti-mouse TMEM26 (1:100, Novus Biologicals) followed by staining with a secondary antibody: Goat anti-rabbit-PE-Cy7 (1:100, Santa Cruz) for 30 min on ice in dark. For analysis of macrophages, cells were stained with antibodies: F4/80-PE (1:100, Abcam), CD11b-BV421 (1:100, BD Biosciences), Hamster mAb against mouse CD11c-FITC (1:100, clone HL3, BD Biosciences), and Rat anti-mouse CD206-Alexa Fluor 647 (1:100, clone C068C2, Biolegend), or their isotype controls (Biolegend) on ice for 30 min in dark. For analysis of ILC2s, cells were stained with lineage cocktail-FITC (1:200, Biolegend), Rat anti-mouse CD5-FITC (1:100, clone 53-7.3, Biolegend), Rat anti-mouse CD45-Pacific BlueTM (1:100, clone 30-F11, Biolegend), Rat mAb against mouse CD25-PE (1:100, clone 3C7, BD Biosciences), and Rabbit antimouse CD127 (1:100, clone 1140A, R&D Systems) followed by staining with a secondary antibody: Goat anti-rabbit-PE-Cy7 (1:100, Santa Cruz), or their isotype controls (Biolegend) on ice for 30 min in dark. After staining, cells were fixed with 2% (w/v) paraformaldehyde and stored at 4 C before analysis with BD LSRFortessa Cell Analyzer (BD Biosciences). Data were analysed using FlowJo software version X.0.7 (Tree Star, Inc.).

Cell Metabolism 26, 1–16.e1–e4, September 5, 2017 e3

Please cite this article in press as: Huang et al., The FGF21-CCL11 Axis Mediates Beiging of White Adipose Tissues by Coupling Sympathetic Nervous System to Type 2 Immunity, Cell Metabolism (2017), http://dx.doi.org/10.1016/j.cmet.2017.08.003

In Vitro Experiments in Primary Adipocytes SVF from scWAT of 12-week-old male C57BL/6J mice was isolated and differentiated into mature adipocytes for 8 days as previously described (Lin et al., 2013). Briefly, SVF was prepared and cultured into DMEM supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific). After reaching confluence, the differentiation cocktail including 0.5 mM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 2 mM rosiglitazone, and 1.7 mM insulin was added to the cells for the first two days, followed by insulin treatment from day 2-8. Eight days after differentiation, more than 80% of cells became mature adipocytes filled with multiple lipid droplets as assessed by oil red O staining. For rmFGF21 treatment, cells were serum starved for 24 hours followed by stimulation with rmFGF21 (1 mg/ml) for another 24 or 48 hours for the detection of mRNA expression levels of Ucp1 and Ccl11 in cell lysates, and UCP1 protein expression in cell lysates and CCL11 protein secretion in conditioned medium. For treatment with chemical inhibitors, cells were serum starved for 24 hours, then pre-incubated with the ERK inhibitors PD98059 (30 mM, Cell Signaling Technology)/U0126 (10 mM, Cell Signaling Technology) or PI3K inhibitor LY294002 (50 mM, Cell Signaling Technology) for 1 hour before treatment with rmFGF21 for another 24 hours for the analysis of mRNA expression of Ccl11 in cell lysates. Western blot, ELISA, and Histological Analyses For Western blot analysis, proteins were extracted from tissues or cells in Radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing a complete protease inhibitor cocktail (Roche), resolved by SDS-PAGE, transferred onto polyvinlidene fluoride (PVDF) membranes (Bio-Rad), and then probed with primary antibodies against UCP1 (Abcam), b-Tubulin (Cell Signaling Technology), and total OXPHOS antibody cocktail (Abcam) followed by incubation with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies. The protein bands were visualized with enhanced chemiluminescence reagents (GE Healthcare) and quantified by using the NIH ImageJ software. CCL11 levels in serum and culture medium and FGF21 levels in serum and tissues were determined by using mouse CCL11/Eotaxin Quantikine ELISA kit (R&D) and mouse FGF-21 Immunoassay kit (Immunodiagnostics Limited), respectively. For ELISA measurements of tissue FGF21, scWAT and eWAT, interscapular BAT and liver from fresh-frozen samples were homogenized in buffer containing 1 M NaCl, 10mM HEPES (pH7.4), and 0.5% Triton X-100 with the cocktail of protease inhibitors (Roche). Cell debris was removed by centrifugation at 12,000 g for 15 min at 4 C. FGF21 protein levels in the protein lysates of different tissues were quantified with the immunoassay as above, and normalized against total protein concentrations. For histological analysis, adipose tissues were fixed in 4% (w/v) paraformaldehyde and embedded in paraffin. Deparaffinized and dehydrated sections were stained with hematoxylin-eosin, or antibodies against mouse UCP1 (Abcam) as previously described (Hui et al., 2015). Tissue sections were visualized and images were captured using an Olympus biological microscope BX41 with DP72 colour digital camera. RNA Extraction and Real-Time PCR Total RNA was extracted from tissues and cells with RNAiso Plus (Takara) and reverse transcribed into complementary DNA with PrimeScript RT reagent kit (Takara). Quantitative real-time PCR was performed by using SYBR Premix Ex Taq (Takara) with specific primers (as listed in Table S1) on a StepOnePlus Real-Time PCR system (Applied Biosystems). Expression levels of all target genes were normalized with the b-Actin gene. For quantification of mitochondrial DNA (mtDNA), total DNA was isolated from adipose tissues using DNeasy 96 Blood & Tissue Kit (Qiagen), and mtDNA content was quantified by real-time PCR with specific primers for the gene CoxII (Primers listed in Table S1). QUANTIFICATION AND STATISTICAL ANALYSIS All analyses were performed with GraphPad Prism 7 (GraphPad, San Diego, CA). Data were presented as mean ± SEM. All experiments were repeated at least three times with representative data shown. Sample size of animal studies (n) was chosen on the basis of literature documentation of similar well-characterized experiments, and no statistical method was used to predetermine sample size. Statistical significance was determined by unpaired two-tailed Student’s t-test and one-way ANOVA for pairwise comparisons or comparisons between multiple groups with single variable, and two-way ANOVA for comparisons with multiple variables. In all statistical comparisons, a p value of < 0.05 was considered as a statistically significant difference. P values, n values, definition of center and precision measures are indicated in the associated figure legends for each figure.

e4 Cell Metabolism 26, 1–16.e1–e4, September 5, 2017